The molecular motor that translocates viral DNA into viral procapsids comprises two components: a terminase that binds and cleaves viral DNA and provides energy for viral DNA packaging, and a specialized portal vertex that engages the terminase and provides the channel in the procapsid shell through which viral DNA is inserted. Several lines of evidence suggest that UL15 UL28 and UL33 encode the herpes simplex virus (HSV) terminase. The first specific aim of this application focuses on a recently discovered DNA binding activity of the purified terminase subunit encoded by pUL33. pUL28 has also been purified in conjunction with pUL33 and, separately a pUL15/pUL28 complex has been purified. These purified components will be tested for binding to DNA sequences known to be involved in cleavage and packaging of viral DNA. The experiments in Aim 2 are designed to determine which components of the terminase interact with the HSV portal vertex. This should shed light on how cleaved DNA is packaged. This will be done by analysis of viral mutants lacking putative portal interaction domains that are defective in DNA cleavage, DNA packaging or both. In this study a newly discovered viral mutant that cleaves, but does not package viral DNA will also be exploited to explore interactions essential for packaging the DNA through the portal vertex into the capsid. This work will have broad medical implications because the terminase and portal are targets of existing novel antiviral drugs and are conserved in all herpesviruses. Thus, the information will have relevance for development of new antiviral drugs with broad specificities to control the wide variety of diseases caused by this important group of pathogens. The third specific aim is based on our discovery that intranuclear capsids move through HSV-infected cell nuclei in an energy- and actindependent manner. Studies are proposed to determine whether this movement is dependent on the HSV packaging motor, a novel actin/myosin motor system, or changes in nuclear morphology induced by HSV infection. The studies in this aim may be important not only for understanding HSV DNA cleavage and packaging, but will likely provide insight into under-explored functions of the nucleus.